Modification of cellular proteins by the small Ubiquitin-like Modifier SUMO is essential for eukaryotic nuclear processes and cell cycle progression in yeast. Unlike ubiquitin conjugation, which generally targets proteins for degradation by the proteasome, SUMO modification targets proteins for changes in activity and localization. SUMO conjugation is catalyzed by at least three enzymes, E1, the SUMO activating enzyme, E2, the SUMO conjugation enzyme, and Ulps, SUMO proteolytic processing and deconjugation enzymes. SUMO is translated as a precursor, requiring C-terminal Ulp proteolysis to generate the mature form. SUMO is activated via C-terminal adenylation by E1 and subsequently transferred to an intramolecular cysteine, forming a thioester bond between SUMO and E1. The E1-SUMO thioester is then transferred to an E2 cysteine forming a thioester bond between E2 and SUMO. The E2-SUMO complex is competent to transfer SUMO to lysine receptors on the protein target, forming a stable isopeptide linkage. SUMO modification is reversible, and its removal from lysine is catalyzed by U1p proteases. Pathways modulated by SUMO conjugation include activation of mammalian RanGAP1, p53 transcriptional regulation, MDM2 ubiquitin ligase regulation, I-kappa-B-alpha protection from ubiquitination, PML, PML-RAR, and SP100 nuclear localization, centromere segregation, and septin ring formation, indicating SUMO conjugation as a central regulator in cellular processes involved in nuclear metabolism and cell cycle control.Since few molecular details are known about the SUMO conjugation pathway, structural, biochemical and genetic techniques will be utilized to establish physical models for the sumoylation process by characterizing E1 activation of SUMO, E2 mediated SUMO conjugation, E2-target protein complexes, and complexes between sumoylated proteins, conjugating, and deconjugating enzymes. Genetic and biochemical models will be established for sumoylation through identification of novel SUMO modified proteins. A thorough structure-function analysis of the SUMO pathway will yield insights into its underlying biology.